ITTO20100611A1 - COMPOSITION OF BIO-REGENERATIVE, REPARATORY AND EUTROFIZZANTE ACTIVITIES - Google Patents

Description

"Composition with bio-regenerative, reparative and eutrophicating activity"

DESCRIPTION

The present invention refers to a composition comprising Polynucleotides (PN) and hyaluronic acid, having healing, re-epithelizing, bio-regenerative and eutrophicating activity against various connective and / or epithelial tissues, including skin and bone tissues. , of the joints, of the osteo-tendon and myo-tendon junctions, of the mucous membranes and of the structures of the visual apparatus, and therefore useful in the treatment of pathologies affecting these tissues, as well as in cosmetics.

Bioregeneration and wound repair have common pathophysiological bases, which classically develop with the sequence:

1. inflammatory phase

2. proliferative phase

3. remodeling phase.

These formally distinct and sequential steps are biologically often substantially overlapping. We can say that in these processes the composition object of the invention acts by favoring the initial phases above all, but not exclusively, by means of Hyaluronic Acid, the final phases above all, but not exclusively, by means of Polynucleotides (PN).

In the inflammatory phase, there is a progressive reaction by cytokines that induce the release of large hyaluronic acid (thousands of kDalton) with a moisturizing function and facilitating cell migration, which then shatters into fragments with a marked pro-inflammatory role facilitating the first reparative phases. The function of hyaluronic acid is therefore characteristic of the first phase of the inflammatory process and is a prelude to the correct development of the "wound-healing" process.

Cell migration of fibroblasts and neoangiogenesis are central phenomena in which the first phase occurs.

In the proliferative phase, the fibroblasts regenerate and produce extracellular matrix. In this phase, the hyaluronic acid is no longer necessary and the "filling" role is ensured by all the components both dermal and various other tissues (collagen, GAG, proteins, glycoproteins, etc.) secreted by the own cells of the affected tissue. This phase is mainly regulated by polynucleotides.

The present invention is based on the recognition of the fact that the joint administration of hyaluronic acid and PN exerts a positive effect in all the phases of regeneration, eutrophication and tissue repair, showing a synergy between the components which allows to obtain results superior to the administration of the components used separately.

The object of the invention is thus a composition with a synergistic effect comprising polynucleotides and hyaluronic acid and its use in the applications defined by the following claims, which form an integral part of the description.

Hyaluronic acid (HA) is the only non-sulfur polymer belonging to the glycosam minoglycan family; It is ubiquitous in mammalian tissues, has no ramifications in its structure and consists of the ordered repetition of a disaccharide composed of glucuronic acid and N-acetylglucosamine.

The size of HA is variable, but exceeds one million Dalton, varying in certain tissues between 3 and 9 million. Initially, it was isolated in 1934 by Mayer in the hyaloid substance of the vitreous body, hence the name, and for years HA has always been considered a filling and structural substance with extraordinary hydrophilic capacities, without a particular biological role. Only in the last decade has we focused on the biological role of this polymer, especially after the discovery of tissue HA receptors, such as CD44 and RHAMM. The knowledge on the role of HA as an active substance in more complex processes concerning tissue regeneration and repair has therefore been extended.

The organization of the data available in the literature has therefore introduced a new concept, in which HA is no longer considered an inert molecule, but actively involved in important biological processes, such as cell migration and duplication, angiogenesis and tissue development and maturation.

The functions of HA change radically according to its size, sometimes producing opposite effects. In fact, the high-weight molecules are highly hydrating and facilitate cell migration while the smaller fragments have an action favoring inflammatory processes. Phenomena that belong to the early stages of the regenerative and reparative processes.

A. Within the scope of the present invention, hyaluronic acid can be used as such or variously salified, for example with sodium.

Typically, hyaluronic acid or sodium salt is used with a molecular weight between 500 and 3000 kDalton, preferably between 600 and 1600 kDalton.

B. The term "Polynucleotides" is intended to indicate a fraction of nucleic acid polymeric chains, preferably DNA, having a molecular weight distribution preferably in the range between 0.5 kDalton and 3000 kDalton, more preferably between 30 kDalton and 2000 kDalton, with a number average molecular weight for example of about 250 kDalton. Said fraction of DNA polymeric chains is preferably obtained by extraction from natural sources, of animal or vegetable origin, such as fish or plant sperm, by means of a process which preferably comprises a partial fragmentation step of the DNA chains, in order to reduce their weights. molecular, and a phase of partial purification of the DNA to eliminate the informational capacity.

The preferred percentage of DNA purification is preferably in the range of 1% to 5% of purine bases removed (ie apurinic sites) on the total purine bases originally present. A preferred purification percentage is between 2% and 2.5%.

From the foregoing, it is evident that the characteristics of PN are significantly different from classical polynucleotides which are not subjected to:

â € ¢ partial purification,

â € ¢ sterilization techniques for viruses and prions â € ¢ purification procedures of the product, which when dry exceeds 98% of purity with quantities of proteins lower than 0.5% and with a molecular weight range that varies from 0, 2 to 3000 K Dalton.

It is known and documented that PNs have the ability to induce mitosis in fibroblasts, endothelial cells and other cells. They have been used to stimulate tissue repair and in many experimental models they have shown a remarkable ability to re-synthesize molecules of the extracellular matrix.

In all wound-healing processes, the scientific literature has confirmed that PNs have a positive effect in repairing damage. PNs also have a remarkable ability to modulate the action of many inflammatory factors and to interact with physiological growth factors, which makes them very important and biologically very active molecules on the growth of different cellular stems and on their secretory activity.

The polynucleotides used have a molecular weight distribution preferably in the range between 0.2 kDalton and 3000 kDalton, more preferably between 30 kDalton and 2000 kDalton, with for example number average molecular weight of about 250 kDalton.

The composition according to the invention comprises: - hyaluronic acid as described above

- polynucleotides (PN) as described above

For liquid formulations (for example injectable formulations or eye drops), polyols are used to obtain suitable osmolarity, preferably mannitol in concentrations from 0 mg / ml to 50 mg / ml, while to ensure the pH around neutrality (7.0 / - 1.0) a buffer system is used, preferably phosphate buffer.

The mannitol to be used is sterile, pyrogenic, pharmaceutical grade, for injectable use in compliance with the European Pharmacopoeia.

The phosphate buffer to be used is pharmaceutical grade, for injectable, sterile, non-pyrogenic use.

Typically, compositions comprising:

- polynucleotides from 1 mg / ml to 20 mg / ml, preferably from 5 mg / ml to 12 mg / ml,

- hyaluronic acid from 1 mg / ml to 20 mg / ml, preferably from 5 mg / ml to 12 mg / ml with polynucleotide / hyaluronic acid ratios by weight, ranging from 0.2: 1 to 2: 1.

Example 1

Mannitol: 40 mg / ml phosphate buffer: 1.3 mg / ml hyaluronic acid: 7.5 mg / ml PN: 7.5 mg / ml Example 2

Mannitol: 40 mg / ml phosphate buffer: 1.3 mg / ml hyaluronic acid: 10 mg / ml PN 10 mg / ml Example 3

Mannitol: 40 mg / ml phosphate buffer: 1.3 mg / ml hyaluronic acid: 3.5 mg / ml PN 3.5 mg / ml Example 4

Mannitol: 40 mg / ml phosphate buffer: 1.3 mg / ml hyaluronic acid: 5 mg / ml polynucleotides: 15 mg / ml Example 5

Mannitol: 40 mg / ml phosphate buffer: 1.3 mg / ml hyaluronic acid: 3 mg / ml PN (respectively): 7.5, 11, 16 mg / ml The synergistic effect of the compositions according to the invention was determined by in vitro tests, designed to determine the effect on cell growth and protein expression of pure hyaluronic acid, pure PN and the combination of the two.

Description of the tests

Human origin fibroblasts were cultured on growth medium (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FCS). The cells were seeded at a density of 10,000 / cm <2>. After 24 hours from seeding the cells were incubated under the conditions reported in the table, always prepared in DMEM 10% FCS.

Cell numbers and protein quantification were performed after 3 days of treatment. The number of cells was measured with a cell counting instrument, protein quantification was performed by spectrophotometric reading of stained protein lysates using a modified procedure of Lowry's method.

The following table shows the concentrations of the tested compositions. The values relating to cell growth and protein content are expressed as% of the control, the latter to be considered as the culture of fibroblasts not subjected to any treatment, measured at the same point-time.

Table 1

COMPOSITION CONCENTRATION GROWTH EXPRESSION (µg / ml) CELL PROTEIN (% with respect to (% with respect to control) to control) Control: 100 100 Hyalu- 12 104 103 Rononic acid (HA) 37 104 105

50 103 104

111 105 104

333 106 107

PN 12 117 115

37 121 119

50 125 126

111 127 127

333 129 128

HA PN 6 6 112 113

18.5 18.5 126 125

25 25 128 128

55.5 55.5 130 131

166.5 166.5 133 131

From the data in table 1 and the related graphs in figure 1 and figure 2, it appears that hyaluronic acid does not change its action as its concentration increases, constantly maintaining a modest effect, both as regards protein expression and growth. cell phone. While it is evident that polynucleotides increase their effect with increasing dose.

Compared to the individual components, the effect obtained from the mixture - which at the same total concentration is made up of 50% hyaluronic acid and 50% polynucleotides - is constantly higher than the effect of PN used alone and naturally of HA used alone (example: the comparison was made between 37 µg / ml of HA used alone, 37 µg / ml of PN used alone and the mixture containing 18.5 µg / ml of each of the two); therefore, bearing in mind the low incidence of hyaluronic acid alone, it is evident that the presence of the latter is important for increasing the effect of PN, which constitute the component that has a real significance on cell growth and on protein expression.

It can be concluded that by combining HA and PN a synergistic effect is obtained at the level of cell regeneration and deposition of the protein matrix.

Taking note of the fact that the concentration of hyaluronic acid is substantially irrelevant for the purposes of cell growth and matrix production, a further test was carried out in which the concentration of hyaluronic acid was kept constant at 1 mg / ml and the PN have been used in increasing concentrations. The choice of setting the concentration of HA at 1 mg / ml was determined by previous experiences in which, by testing only the HA at various dilutions, it was seen that, despite being relatively constant and modest its ability to stimulate cell regeneration and secretion, this was the concentration of maximum efficacy under the experimental conditions.

The choice of setting the concentration of Hyaluronic Acid to be used in the mixture at 1 mg / ml was determined by the data obtained by testing Hyaluronic Acid at different dilutions on cultured fibroblasts for 3 and 7 days. The evaluation of cell growth was carried out using the Alamar Assay method. The data are summarized in the graph of figure 3, expressed as a percentage of growth of the treated cells with respect to the control set equal to 100.

On the basis of these experimental data, the dose of 1 mg / ml of HA was isolated as the most suitable dose as it is the maximum that can be administered without negatively affecting cell growth, thus making the most of the hydrating effect of HA.

The graph in figure 4 and the related table 2 below confirm that HA significantly enhances the effect of PN, in fact 12 µ / ml of PN in the presence of HA obtain results equivalent to 100 µg / ml in the absence of HA .

TABLE 2

PN PN + HA

mean sd mean ds min max min max 0 100 3.9 112.8 8.14 96.1 103.9 104.66 120.94 12 117 4.6 126.82 9.45 112.4 121.6 117, 37 136.27 100 127.8 4.88 133.03 11.4 122.92 132.68 121.63 144.43 Moreover, HA is a substance that plays a decisive role in regulating tissue hydration and this is an important advantage which adds up to the enhancement effect on PN, adding to the mixture an effect that is undoubtedly useful for the uses which form the subject of the invention.

Therefore, the combination of polynucleotides and hyaluronic acid in the same mixture has a complementary effect: the former stimulate the increase of fibroblasts and extracellular protein content, the latter favors the hydration of the matrix. In this way a synergistic activity is achieved which by restoring all the components of the treated tissue allows the recovery of tissue trophism, where necessary, the repair of lesions with loss of substance.

In view of the observed results, the compositions according to the invention can be used in all applications of a therapeutic or cosmetic nature in which the following activities are required:

a) skin bio-regenerative activity, for example for the face, neck, décolleté, skin in general, skin folds and mucous membranes (large and small lips) and skin appendages, such as the scalp for biorevitalization of the hair;

b) trophic, reparative and lubricating activity, both in topical application (lesions or dystrophies of the skin), and in application through intra-articular infiltration;

c) trophic and reparative activity through periwound infiltrations in case of ulcers, decubitus and loss of substance of different nature;

d) re-epithelialization of the conjunctiva and cornea, in topical administration using eye drops, creams or ophthalmic gels.

The pharmaceutical forms suitable for the above applications can be:

- multipurpose bottles, single use ampoules, pre-filled and disposable syringes,

- sprays or foams, gauze or other medicated supports for topical applications,

- liquids or emulsions or other forms for transdermal administration using suitable equipment,

- mixtures with excipients suitable for formulations that can be administered on mucous surfaces. natural orifices (oral cavity, vagina, rectum): ovules, glow plugs, vaginal tablets and tablets, cannulas, ointments, creams, gels,

- ophthalmic drops, as a substitute for tears and / or with a restorative effect,

- emulsions (creams, ointments, serums, gels) of various types for topical applications both for aesthetic / regenerative purposes, and for activities favoring the repair of loss of substance, intended for epithelia, mucous membranes and connective tissues, including ocular structures.

Claims (17)

CLAIMS 1. Composition comprising hyaluronic acid and a mixture of polynucleotides, extracted from natural sources, for use in the therapeutic and / or cosmetic treatment of regeneration and / or repair of connective and / or epithelial tissues, including skin tissues, bone, joints, osteo-tendon and myo-tendon junctions, mucous membranes and structures of the visual apparatus. 2. Composition comprising hyaluronic acid and a mixture of polynucleotides from a natural source, for the therapeutic and cosmetic bio-regeneration treatment of the skin or scalp. 3. Composition comprising hyaluronic acid and a mixture of polynucleotides from natural sources for use in repair treatments of skin tissue and osteo-tendon and myotendinous junctions by subcutaneous infiltration, or by periwound infiltration. 4. Composition comprising hyaluronic acid and a mixture of polynucleotides from a natural source for use in treatments of joint pathologies by means of intra-articular infiltrations. 5. Composition comprising hyaluronic acid and a mixture of polynucleotides from a natural source for use in re-epithelializing and / or wetting treatments of the conjunctival epithelium. 6. Composition comprising hyaluronic acid and a mixture of polynucleotides from natural sources for cutaneous use in formulations suitable for transdermal transport using suitable equipment. 7. Composition according to any one of claims 1 to 6, comprising from 1 mg / ml to 20 mg / ml of hyaluronic acid and from 1 mg / ml to 20 mg / ml of said mixture of polynucleotides. 8. Composition according to claim 7, comprising from 5 mg / ml to 12 mg / ml of hyaluronic acid and from 5 mg / ml to 12 mg / ml of said mixture of polynucleotides. 9. Composition according to claims 7 or 8, comprising polynucleotides and hyaluronic acid in a weight ratio ranging from 0.2: 1 to 2: 1. 10. Composition according to any one of the preceding claims, wherein said mixture of polynucleotides is partially purified. 11. Composition according to claim 10, wherein said mixture of polynucleotides has a percentage of purification comprised between 1% and 5% of purine bases removed on the total of purine bases originally present. Composition according to any one of the preceding claims, wherein said mixture of polynucleotides is a fraction of nucleic acid polymeric chains having a molecular weight distribution comprised between 0.5 kDalton and 3000 kDalton, or preferably between 30 kDalton and 2000 kDalton , with for example an average molecular weight of about 250 kDalton. 13. Composition according to any one of the preceding claims, wherein said mixture of polynucleotides can be obtained by extraction from a natural source, animal or vegetable, by means of a process comprising a partial fragmentation step of the polynucleotide chains of nucleic acid and a partial purification step of nucleic acid. 14. Composition according to any one of the preceding claims, comprising hyaluronic acid with a molecular weight between 500 kDalton and 3000 kDalton. 15. Composition according to any one of the preceding claims in liquid form, free of physiological saline solution, in an aqueous solvent comprising a pharmaceutically acceptable polyol, preferably mannitol. 16. Composition according to claim 15, having an osmolarity between 10 m.o.s./l and 500 m.o.s./l, preferably between 250 m.o.s./l and 350 m.o.s./l. 17. Composition according to any one of the preceding claims, in dosage form suitable for topical application or for intra-articular, subcutaneous or periwound injection / infiltration.